Without limiting the scope of the invention, its background is described in connection with protein glycosylation.
The nature of N-glycosylation attached to therapeutic proteins is a critical attribute of the protein identity as it can affect its therapeutic activity, stability and immunogenicity properties. The N-glycan profile of recombinant proteins depends on the host used for its production and the host culture conditions. There is an increasing demand for manufacturing processes leading to the production of homogeneous and consistent therapeutic glycoproteins, composed ideally of one single optimal glycoform. For instance, afucosylated monoclonal antibodies have increased cytotoxicity activity. Also, human serum proteins used as therapeutics often require human-specific sialylation, which remains a challenge to uniformly produce in heterologous expression systems.
An in vitro therapeutics glyco-remodeling technology has been proposed by others to meet the demand of glycoengineered therapeutics (Huang, W., Giddens, J., Fan, S., Toonstra, C., & Wang, L. (2012). Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. Journal of the American Chemical Society, 134, 12308-12318; and Wang, L., & Lomino, J. V. (2012). Emerging technologies for making glycan-defined glycoproteins. ACS chemical biology, 7(1), 110-22). The methods taught require the extraction and purification of the glycoprotein of interest, the deglycosylation of the protein, in such manner that the first N-acetylglucosamine (GlcNAc) and fucose, if attached remain attached to the asparagine residue of the protein, and the reglycosylation of the protein with a purified or synthesized activated N-glycan donor of choice using a proprietary endoglycosidase S (EndoS) mutant. The deglycosylation and reglycosylation steps are preceded and followed by purification steps that render the glycoremodeling process laborious and costly (See FIG. 1, Option A, and FIG. 2A, labeled as prior art).
Glycoremodeling methods are taught in, e.g., U.S. Pat. Nos. 8,361,961; 7,956,032; 7,696,163; and 7,338,933, issued to DeFrees, et al., include methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide, O-linked glycosylation of peptides, and glycopegylation of proteins.
Another method is taught in United States Patent Application No. 20130137857, filed by Wang, et al., entitled, Transglycosylation Activity Of Glycosynthase Mutants Of An Endo-Beta-N-Acetylglucosaminidase (Endo-D) From Streptococcus Pneumoniae. Briefly, the invention is said to include recombinant Endo-D and selected mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor by transglycosylation. Such recombinant Endo-D and selected mutants are said to be useful for efficient glycosylation remodeling of IgG1-Fc domain.
Yet another method is taught in United States Patent Application No. 20100173323, filed by Strome, et al., entitled Glycosylation Engineered Antibody Therapy. Briefly, this application is said to teach methods of generating a glycosylation-engineered antibody, and using the glycosylation-engineered antibody for treating a patient, particularly a cancer patient or a patient with an immune disease or disorder. The invention also includes methods of generating a glycosylation-engineered antibody for use in the treatment of patients having a polymorphism that does not respond to conventional antibody therapy, methods of improving the biological activity of an antibody by glycosylation engineering, and methods of modulating antibody-dependent cell-mediated cytoxicity (ADCC) using a glycosylation-engineered antibody.